Protein biogenesis in the secretory pathway involves several processes that overlap in time with polypeptide chain synthesis. First, the nascent polypeptide chain must be targeted correctly to the membrane of the endoplasmic reticulum (ER). Then, its translocation to the lumen of the ER is initiated. Folding of the polypeptide chain has to begin early in its translocation. During and immediately after translocation, posttranslational modifications occur, and decisions are made by the cell regarding degradation of undesired chains, including translocation back to the cytoplasm and degradation by the proteasome. Of the processes contemporaneous with translocation, perhaps the most profound is protein folding, because it is a crucial step in the decoding of the information in the genome. A misfolded protein may be as bad as, or worse, than not having the protein at all. If proteins have multiple folded states with distinct functions, the precise pathway of folding and its regulation will determine which function is actually expressed, and to what extent.
A fundamental dogma of modern biology is that primary structure determines secondary structure, which together with appropriate post-translational modifications such as disulfide bond formation, determines the tertiary (and quaternary) protein structures. This organization, from primary structure secondary structure tertiary structure, constitutes a “first order” organizing principle for protein folding. Implicit in the term “structure” is the notion or a unique, stable entity; but protein structure is a statistical concept. Native protein conformations are energetically preferred relative to unfolded, denatured forms of the same chains; but the energetic preference is modest (10 kCal/mole), which means that proteins are dynamic, fluctuating entities, and even “stable” proteins will unfold, to some degree, transiently.
Translocation overlaps with protein folding, therefore one would expect that, in the course of evolution, these two processes would have influenced one another. Folding pathways may have been modified to accommodate the needs of translocation; translocation pathways may have been modified to accommodate the needs of folding. A growing body of literature provides support for both of these possibilities. The most dramatic example to date of translocational regulation, with implications for folding, is seen in the biogenesis of the prion protein (PrP). In the case of PrP, a homogeneous population of nascent chains results in three topological forms. One of them, secPrP, appears to be fully translocated (secreted) across the ER membrane and tethered by a C-terminal glycolipid anchor; this is the form observed in normal brain. Although the function of secPrP is unknown, it seems likely, by analogy to other glycolipid anchored proteins, to have signaling functions in the nervous system. A recently demonstrated anti-apoptotic function appears consistent with this role. The other two forms of PrP span the membrane once in opposite orientations, with a membrane-spanning stretch at approximately amino acids 112±30. By contrast, the other two forms of PrP are made as singly-spanning membrane proteins in opposite orientations with either the N- or C-terminus in the ER lumen (termed NtmPrP and CtmPrP, respectively). CtmPrP triggers spontaneous neurodegeneration when overexpressed. Furthermore, in infectious prion disease, CtmPrP appears to be induced just prior to onset of clinical signs, suggesting that it initiates a final common pathway to neurodegeneration. Other studies implicate an as yet unknown glycoprotein of the ER membrane as a translocation accessory factor (TrAF) that “protects” the normal brain from expression of CtmPrP by directing nascent PrP chains to the pathway leading to SecPrP.
The distinction between SecPrP and CtmPrP is usually made on topological grounds. However, these two polypeptides of identical sequence also differ in their conformation. This was demonstrated by their differential sensitivity to limited protease digestion in non-denaturing detergent solutions. Thus, translocational regulation appears to be a means of generating multiple forms of PrP that differ in both conformation and function. The machinery (i.e. a TrAF) that directs nascent PrP chains to make SecPrP rather than CtmPrP, may itself be regulated, based on the ability of scrapie infection to increase the amount of CtmPrP detectable in brain. Although PrP is currently the best example of translocational regulation, there is evidence for similar principles being utilized by a broader set of proteins. Together, these observations lead to a new principle: a protein's conformation is determined not just by its primary amino acid sequence, but also by proteins such as TrAF, that influence which of two or more different functional conformational outcomes actually occur or predominate.
It therefore is of interest to determine if complex secretory or integral membrane proteins can have multiple distinct functional folded states. At present, the major limitation to testing these and other hypotheses of conformational control is a lack of tools to recognize the heterogeneity of functional protein conformations. But for the fortuitous coincidence that, in PrP, conformational heterogeneity was expressed as topological heterogeneity, it might not yet have been detected. Better tools such as panels of conformation-specific monoclonal antibodies that would allow relative reactivities of subpopulations of newly synthesized proteins to be scored, and thereby define conformational differences are needed. This approach would allow a catalogue of the conformational states utilized by a given protein in health and during the progression to disease as well as a means for inhibiting the disease.